Intestinal proteases play an important role in digestion and inflammatory response. Dysregulated proteolytic activity has been associated with disease states including irritable bowel syndrome (IBS). My ongoing K23 studies mechanisms of post-infection IBS (PI-IBS) following Campylobacter enteritis, focusing on the role of intestinal barrier function and microbiota. Data from that project revealed that fecal proteolytic activity is increased in 40% of PI-IBS patients. This subset with high fecal proteolytic activity has greater colonic in vivo and mucosal permeability as compared to those with low proteolytic activity. The effect on the intestinal barrier function was inhibited by serine protease inhibitor and was at least partially mediated by cleavage of protease activated receptor 2 (PAR-2). Another key observation we made is that transfer of human microbiota into germ-free mice results in significant inhibition of fecal proteolytic activity, suggesting a novel mechanism for commensal microbiota to affect host physiology. Humanization with microbiota from high proteolytic activity PI-IBS patient results in ineffective inhibition, as compared to low proteolytic activity PI-IBS patient or healthy volunteer. This R03 application's proposed aims are based on strong preliminary data generated from the ongoing K23, will allow developing the rationale and provide additional preliminary data for an R01 application in this area. The overall hypothesis of this R03 proposal is that serine protease mediated activation of PAR disrupts barrier function in PI-IBS patients in a microbiota dependent manner. Specific Aim 1 is to determine the mechanism by which serine proteases disrupt barrier function in PI-IBS. This aim's underlying hypothesis is that serine proteases disrupt barrier function in PI-IBS patients through PAR cleavage in an activity-dependent manner. We plan to examine if pore, leak, and unrestricted pathways of the barrier function are involved. Secondly, we will determine affected barrier function pathways in PI-IBS patients with high proteolytic activity. We will also develop crypt-derived colonoid monolayers and determine if they can be used as host-specific models of barrier function. This aim will utilize fecal supernatants and biopsies derived from PI-IBS patients with two different ranges of high proteolytic activity and low proteolytic activity controls. Specific Aim 2 is to determine the proteases and protease inhibitors responsible for elevated fecal proteolytic activity in PI-IBS. We hypothesize that elevation of specific serine proteases in PI-IBS patients is due to their ineffective suppression by microbiota derived protease inhibitors. We will use metaproteomics to determine peptide and protein sequences in the fecal supernatants from high and low proteolytic activity PI-IBS patients. Their origin (host or bacterial) will be determined. Additionally, we will utilize zymography to test protease inhibitory properties of fecal supernatants from patients who have low fecal proteolytic activity. Overall, these two aims will help understand the role of luminal proteolytic activity on intestinal barrier function and characterize the proteases and protease inhibitors in the fecal supernatants from PI-IBS patients. These experiments will provide targets that can be tested for their protease inhibitory properties.